Figure 1
Figure 1. Chronic exposure to LPS leads to T-cell dysfunction associated with abnormal ζ-chain expression in mice with a functional TLR4. (A) Mice were treated 3 times with MLVs encapsulated with LPS as described in “Antigen, TLRLs, and immunization.” The mice were killed 2 days after the last treatment. (B) Splenocytes from control C3H/HeN (I,IV), LPS-treated C3H/HeN (II,V), and C3H/HeJ (III,VI) mice were labeled with CFSE and then activated ex vivo with anti-CD3 and anti-CD28 antibodies or with PMA and Ca2+ ionophore as described in “6-Carboxyfluorescein succinimidyl ester staining and ex vivo proliferation assay” (black histograms) or left untreated (white histograms). The proliferative response was assessed by monitoring cell divisions of gated CFSE-labeled Thy1.2+ T cells. (C) Splenocytes from control and LPS-treated mice were activated for 48 hours and specific T cell proliferation was measured by BrdU incorporation in Thy-1.2+ cells using FACS. The results are presented as the mean value of 3 independent experiments, and standard deviations are shown. *P < .02; **P < .001 (Student t test). (D) Splenic Thy1.2+ T cells from LPS-treated (black line) and control (gray area) C3H/HeN mice (I,II) or C3H/HeJ mice (III,IV) were analyzed for total ζ (I,III) and CD3ε (II,IV) expression levels by FACS. (E) Equal numbers of splenic T cells from LPS-treated and control mice were lysed and subjected to Western blot analysis. Expression of the protein tyrosine kinase ZAP-70, CD3ε and ζ-chain was assessed by immunoblotting (IB) using specific antibodies. A representative experiment is shown of 5 performed.

Chronic exposure to LPS leads to T-cell dysfunction associated with abnormal ζ-chain expression in mice with a functional TLR4. (A) Mice were treated 3 times with MLVs encapsulated with LPS as described in “Antigen, TLRLs, and immunization.” The mice were killed 2 days after the last treatment. (B) Splenocytes from control C3H/HeN (I,IV), LPS-treated C3H/HeN (II,V), and C3H/HeJ (III,VI) mice were labeled with CFSE and then activated ex vivo with anti-CD3 and anti-CD28 antibodies or with PMA and Ca2+ ionophore as described in “6-Carboxyfluorescein succinimidyl ester staining and ex vivo proliferation assay” (black histograms) or left untreated (white histograms). The proliferative response was assessed by monitoring cell divisions of gated CFSE-labeled Thy1.2+ T cells. (C) Splenocytes from control and LPS-treated mice were activated for 48 hours and specific T cell proliferation was measured by BrdU incorporation in Thy-1.2+ cells using FACS. The results are presented as the mean value of 3 independent experiments, and standard deviations are shown. *P < .02; **P < .001 (Student t test). (D) Splenic Thy1.2+ T cells from LPS-treated (black line) and control (gray area) C3H/HeN mice (I,II) or C3H/HeJ mice (III,IV) were analyzed for total ζ (I,III) and CD3ε (II,IV) expression levels by FACS. (E) Equal numbers of splenic T cells from LPS-treated and control mice were lysed and subjected to Western blot analysis. Expression of the protein tyrosine kinase ZAP-70, CD3ε and ζ-chain was assessed by immunoblotting (IB) using specific antibodies. A representative experiment is shown of 5 performed.

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